Complex traits and diseases tend to cluster in families, suggesting the contribution of genetic factors. Although hundreds of genetic variants have been associated with complex diseases, most confer modest disease risk and fail to explain the observed heritability. The search for the sources of this ?missing heritability? is ongoing. We propose that copy number variation in ribosomal RNA genes may be an underlying, yet uncharacterized, genetic cause of heritable phenotypic variation, and hence a source of missing heritability. Ribosomal RNA genes (rDNA) exist as tandem gene arrays in all eukaryotes. The repetitive nature of these arrays predisposes them to instability and thus substantial copy number variation among individuals. In preliminary data, we find vast variation in rDNA copy number among both wild C. elegans isolates and nominally isogenic laboratory worms, which correlates with lifespan, mutation penetrance, and protein homeostasis. We have shown previously that variation in rDNA dosage is associated with variation in global gene expression in flies and humans. Despite its extraordinary importance for cellular physiology, rDNA and its copy number variation are grievously understudied, due to the technical challenges of genotyping repetitive DNA, and have heretofore been ignored in genome-wide association studies (GWAS). To overcome these technical challenges, we have developed a single-molecule-enabled capture technology that accurately counts rDNA copy number at low cost and high throughput. We will apply this technology to two model organisms, Caenorhabditis elegans and Drosophila melanogaster, which are each uniquely suited to address our hypothesis that rDNA copy number associates with complex traits. C. elegans is the model of choice for studying complex traits such as lifespan and stress response and allows for automated, high- throughput phenotypic sorting. D. melanogaster is the preeminent model for complex trait genetics, offering numerous, previously phenotyped lines in the Drosophila melanogaster Genetics Reference Panel (DGRP) as a GWAS resource. In Aim 1, we will adapt our technology to genotype rDNA copy number in flies and human cells. In Aim 2, we will test for correlations of rDNA copy number with disease-relevant complex traits, including lifespan, stress response, mutation penetrance, and protein homeostasis, across otherwise isogenic C. elegans populations. To test causality of rDNA genotype for phenotype, we will engineer rDNA copy number changes in C. elegans and characterize the resulting phenotypic consequences. In Aim 3, we will determine to what extent rDNA copy number variation contributes to phenotypic variation, either additively or epistatically, across the 205 DGRP lines and whether inclusion of rDNA copy number reduces missing heritability. The tested traits will include whole-genome gene expression, sleep traits, lifespan, alcohol sensitivity, male aggression, and starvation resistance, among others. Further, we will characterize the range of rDNA copy number variation among individual flies within DGRP lines and assess the inheritance and mutability of rDNA in defined pedigrees. In sum, we will explore a currently uncharacterized class of genetic variation with potentially vast phenotypic impact and develop technology that makes this genetic variation accessible for future GWAS in humans.